We have expressed in E. coli and purified a native protease precursor (TF-PR) of the human immunodeficiency virus, type 1, that contains the protease (PR) coding sequence linked to 60 residues of the native flanking trans-frame (TF; otherwise termed p6*) region at the N-terminus and investigated the kinetics of its maturation to release the mature protease. Upon renaturation, the TF-PR which possesses very low enzymatic activity undergoes time dependent autoprocessing to release the mature protease concomitant with an increase in enzymatic activity. Initial rates monitored by following the increase in enzymatic activity are linearly dependent on protein concentration, indicating that the N- terminal cleavage is first-order in protein concentration. The rate of disappearance of TF-PR and that of the appearance of the mature protease and enzymatic activity are identical; k=0.13 + 0.01 min-1, in 50 mM acetate at pH 4.8. A complete pH-rate profile for the autoprocessing reaction showed that cleavage of ht N-terminal trans-frame region to release the mature protease is dependent on the ionization of groups with pKa's of 4.9 and 5.1. The TF-PR fusion protein is ca. 8-fold more sensitive to urea denaturation as compared to the mature protease suggesting that the presence of the TF sequence linked to the protease domain lowers the stability of TF-PR dimer. Kinetics of the autoprocessing of the TF-PR is consistent with previously published results for the autoprocessing of the model precursor MBP-deltaTF-PR- deltaP0l (Louis et al. Proc. Natl Acad. Sci USA 1994, 91, 7970-7974) providing direct evidence that the intramolecular cleavage that occurs at the N-terminus of protease is independent of the length of sequence flanking the N-terminus of the protease. We prove the presence of a protease cleavage site 8 residues downstream from the mature nucleocapsid protein in the transframe region of the Gag- P0l polyprotein using polyprotein and synthetic peptides as substrates.